Title: Freshwater alien acuta (Draparnaud, 1805) - a possible model for bioaccumulation of heavy metals

Author: Aneta Spyra, Anna Cieplok, Małgorzata Strzelec, Agnieszka Babczyńska

Citation style: Spyra Aneta, Cieplok Anna, Strzelec Małgorzata, Babczyńska Agnieszka. (2019). Freshwater alien species (Draparnaud, 1805) - a possible model for bioaccumulation of heavy metals. "Ecotoxicology and Environmental Safety" (Vol. 185 (2019), art. no. 109703), doi 10.1016/j.ecoenv.2019.109703

Ecotoxicology and Environmental Safety 185 (2019) 109703

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Ecotoxicology and Environmental Safety

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Freshwater alien species Physella acuta (Draparnaud, 1805) - A possible model for bioaccumulation of heavy metals T

∗ Aneta Spyraa, , Anna Cieploka,Małgorzata Strzeleca, Agnieszka Babczyńskab a Department of Hydrobiology, Faculty of Biology & Environmental Protection, University of Silesia, Bankowa 9, 40-007, Katowice, Poland b Department of Physiology and Ecotoxicology & Environmental Protection, University of Silesia, Bankowa 9, 40-007, Katowice, Poland

ARTICLE INFO ABSTRACT

Keywords: In this study we focused on Physella acuta, an alien snail species in order to determine their ability of bioac- Heavy metal pollution cumulation of heavy metals in their shells, bodies, the difference in accumulation in relation to age classes, and Freshwater snails the influence of ecological variables on the community composition and density. On the basis of the results of Alien species ecological, toxicological, and experimental analyses we aimed to study the potential invasive features of P. acuta BAF index in comparision with the native species palustris. The content of Cu and Zn in the substratum and Accumulation ammonia in the water was strongly related to the patterns of distribution of P. acuta. The content of Cd, Pb, and Cu in the shell fraction was always significantly lower than in the body fraction. A comparison of accumulation with respect to the size classes of P. acuta indicated that the lowest metal concentration in the body was typical for the largest individuals, except for Zn. Metal content in the bodies of the native species did not differ from the content measured in their analogous group of the largest individuals of P. acuta. The lowest value of bioaccu- mulation factor (BAF) was found for the large class of specimens of this species for each metal. A distinct decrease in the value of BAF in relation to the size of snails was found for cadmium. A 100% hatching success found in masses collected from pond confirmed the high reproductive potential of P. acuta which can be a factor that promotes its invasive features following its ability to occur in very high densities, but not necessarily the ability of metal accumulation in the body. Physella acuta can be used as a model organism in the studies on the accumulation of heavy metals however, the extend of accumulation can differ among the age classes. Because of the high tolerance of P. acuta to heavy metal pollution, in the future this species can be found in significantly polluted habitats, inhabiting free ecological niches, and occurring in high densities in snail communities.

1. Introduction as well as in relation to the age class or size. Depending on the en- vironmental conditions (different values of pH and total hardness, se- Heavy metal pollution of freshwater environments is a subject of diments), their capabilities of metal uptake are differ (Karadede-Akin serious international concern since metals enter the food chain and can and Ünlü, 2007). Several investigations (Ibrahim, 2006; Moolman et al., undergo bioaccumulation, thereby endangering human health (Hossain 2007; Mahmoud and Abu Taleb, 2013) have confirmed that snails are and Aditya, 2013). Industrial emissions and the inappropriate man- suitable organisms for the identification of contaminated sites due to agement of waste waters lead to the continuous pollution of the en- their ability to accumulate large quantities of metals, which is reflected vironment, and thus adversely affect the development of organisms in the values of bioaccumulation factor (BAF). (Cortet et al., 1999; Järup, 2003) and promote the accumulation of BAF belongs to a group of measures that are often used to assess the heavy metals (Du et al., 2011; Lefcort et al., 2015). risks caused by pollutants in relation to the safety of and The mollusc species demonstrate different preferences for the up- human in the event of exposure to the chemicals present in the en- take of various metals (Lefcort et al., 2015) and have been used to test vironment. The factor compares the concentration of a pollutant in the the toxicity of pesticides (e.g. insecticides, and other contaminants food and/or medium (water or soil) and in the animal body and is (PHBs) (Wilbrink et al., 1992; Woin and Bronmark, 1992). These or- calculated as the quotient of the chemical concentration in the body of ganisms exhibit differences in the accumulation of heavy metals in their the animal (or plant) and in its food (or medium) (Arnot and Gobas, body and shell (Khangarot and Ray, 1988; Petare and Waykar, 2018), 2006). According to this factor, environmental scientists often classify

∗ Corresponding author. E-mail address: [email protected] (A. Spyra). https://doi.org/10.1016/j.ecoenv.2019.109703 Received 13 June 2019; Received in revised form 18 September 2019; Accepted 19 September 2019 0147-6513/ © 2019 The Authors. Published by Elsevier Inc. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/BY-NC-ND/4.0/). A. Spyra, et al. Ecotoxicology and Environmental Safety 185 (2019) 109703 the animals into three groups: macro-, micro- or deconcentrators of a 2. Methods chemical substances if BAF > 2, 1 < BAF < 2 or BAF < 1, respectively (Dallinger, 1993). Both the elimination (as is in the case of deconcen- 2.1. Analytical procedures and bioaccumulation analysis trators) and the safe storage and neutralization (macroconcentrators) of potentially harmful chemicals require energy allocation and a trade-off For this study, Physella acuta and Stagnicola palustris were collected between detoxification and growth, development or reproduction of an from exposed environments and their body and shell were subjected to organism (Shirley and Sibly, 1999; Zhang et al., 2015). The efficiency of chemical analyses to determine the bioaccumulation of heavy metals. the trade-off may contribute to the potential invasiveness of an alien As the native species, S. palustris was selected because it is found in a species colonizing the anthropogenic environments. A study on the higher density than other snails and its size is similar to that of the alien terrestrial invertebrates, ants, indicated that environmental toxins may P. acuta. limit the developmental potential of both native and In the laboratory the collected snails were placed into glass aquaria (De La Riva and Trumble, 2016). It is also stressed that plasticity, in- containing filtered pond water for maintenance for 24 h. Following the cluding the tolerance to pollutants, may enhance the invasiveness of an removal of detritus and decomposing plants collected in the process, the alien species (Novo et al., 2015). This was proven by Matthews et al. snail populations (S. palustris and P. acuta) were segregated into dif- (2015) in the quagga mussel, a species that gained dominance over the ferent size classes based on the shell length (in millimeter) with an zebra mussel. The estimation of metal concentrations in the soft tissues accuracy of 0.5 mm. For each snail, the shell length (from the apex to of both mussel species indicated that the concentrations were higher in the tip of the last whorl in mm) and shell width (width of the last whorl) the invasive quagga mussels than in the zebra mussel individuals. were measured. Snails were classified into three different size classes as Matthews et al. (2015) did not explain the possible mechanisms un- follows: small specimens (juveniles), 1.0–6.9 mm (N = 20), medium derlying this difference but pointed out its ecological consequences. We specimens, 7.0–8.9 mm (N = 25) and large specimens (adults) cannot exclude the possibility that a tolerance to metals may contribute 9.0–12.4 mm (N = 25). We used the same size separation scheme for to the colonizing success of alien species in anthropogenically polluted the native species. The large snail specimens of both species were used environments. to compare bioaccumulation. Physella () acuta (Draparnaud, 1805) is an aquatic pulmonate The concentration of heavy metals (Cu, Pb, Cd, Zn) was analyzed in snail originating from North America, which is notorious for its high the snail samples (soft tissues and shells), leaf deposits, plant deposits, invasive potential (Vinarski, 2017) and called as invasive in some re- and bottom sediments. Plant materials (leaves, plants) and sediments search (Hossain and Aditya, 2013; Van Leuven et al., 2013; Saha et al., were sampled qualitatively using a frame net and bottom scraper. The 2016). It is known to be a highly successful invader worldwide collected materials were placed into plastic containers and were (Holomuzki and Biggs, 2012; Ebbs et al., 2018). Its invasive features are transported to the laboratory. For analyzing the metal concentrations, not so obvious in all the sites of its occurrence for example in Poland, it the snails were anesthetized on ice and dissected to obtain the shells has the status of an alien species but not an invasive one. The very high and body samples. These samples as well as the samples of leaves and expansiveness of this species permits its wide distribution on various plant remnants were dried by heating (approximately- 50 °C) to a continents probably by accidental dispersal mediated by humans during constant mass, weighed to get the dry mass of the sample, slightly the transport of exotic plants to European botanical gardens (Vinarski, crushed and mineralized in quartz tubes in nitric acid (approx. 65%, 2017). Their wide distribution could have also been caused by natural Suprapur ®, Merck KGaA Darmstadt, Germany 0.5 ml of the acid was processes such as long-distance dispersal from the Americas to Europe. used per 50 μg of the sample) at 150 °C until the samples was light straw Our study was focused on snails due to their high rates of metal coloured and perfectly transparent. Then the samples were dissolved in accumulation, responsiveness to changes in metal exposure (Adewunmi 4 mL redistilled water. The digests were analyzed with a et al., 1996), and different sensitivities to metal contaminants (Lefcort ThermoScientific ICE 3500 atomic absorption spectrophotometer in a et al., 2015). They are widely distributed in nearly all types of water graphite furnace for the Pb, Cd and Cu content and in an air–acetylene systems and are frequently very abundant (Elder and Collins, 1991). flame for determining the Zn content. The metal level was calculated Pulmonate species of snails such as P. acuta and Stagnicola palustris are based on similarly prepared Merck standards. The accuracy of the de- known and important for their abundant biomass in the littoral zone of termination was checked using spiked samples with BRC-158 bovine freshwater bodies (Ravera and Mariani, 1966). As was reflected by liver as the reference material (IRMM Geel, Belgium). The percentage Elder and Collins (1991), while some processes of toxicity have been recovery of the spiked samples was high (93– 96%) for the measured studied in marine and freshwater animals they have been, rarely, if concentration of metals. The metal concentrations were expressed as μg ever, demonstrated in alien freshwater snails along with the total en- Me/g dry mass of each sample. Samples of each kind of material (snail vironment (e.g. sediments, substrates, environmental infl uence and shells and bodies, plants) were prepared in six replicates. Mean values ecological importance). were calculated using the results of measurements of six replicates for The purpose of this study was to investigate the potential success of each sample. P. acuta to colonize a new environment by measuring the metal con- BAF was calculated for each metal separately according to the fol- centrations and other ecological parameters. The study also aimed to lowing equation: BAF = Me snail conc/Me plant conc, where: Me snail determine whether the alien snail species differ from the native species conc = mean (shell Me conc + soft tissue conc), Me plant conc = mean in their strategies of metal accumulation/elimination by analyzing the (leaf conc + Typha conc). Snails were classified into three groups as metal deposits in the shell and body and calculating the BAF of the deconcentrators, microconcentrators or macroconcentrators if BAF < 1, tested metals, and whether the size class of the alien species is sig- 1 < BAF < 2 or BAF > 2, respectively. nificant in metal accumulation. Furthermore, the study estimated the reproductive potential of an alien species, which is measured as 2.2. Experimental study hatching success. Additional information on the densities and dom- inance patterns and the probable influence of the environmental vari- For the experimental part of this study, specimens of P. acuta and S. ables on the occurrence of snails are also given. Considering two sites as palustris were collected from an anthropogenic forest pond to determine a source of food with different metal levels we used the alien species as their eggs hatching success. The snails were successively collected a possible model for bioaccumulation and compared this parameter during the period from 2016 to 2017 using qualitative hydrobiological with that of the native species. methods (bottom scraper) together with plants and leaves from the trees. After collection, the snails were separated from the plant mate- rials. Fifty individuals of each species were selected and transported to

2 A. Spyra, et al. Ecotoxicology and Environmental Safety 185 (2019) 109703 the laboratory where they were placed in aquaria under the same the comparison of the means, an inferential statistic - the Levene's test conditions of water and temperature until they laid eggs. In addition, was used to assess the equality of variances. To determine the sig- the egg masses of P. acuta laid in the field were also collected. They nificant differences between the group means in the ANOVA, Scheffe's were found at the surface of the water plant remains as well as on the test, which is considered to be one of the most conservative post hoc leaves. All the masses (LM – laboratory masses, FM – field masses) were tests (Winer et al., 1991), was adopted. measured, and the eggs in the masses were counted. None masses of For the analyses of metal concentrations, the data were tested for Stagnicola were observed in the field during the study period, or during normality using the Kolmogorov–Smirnov and Lilliefors tests, and then the experimental study. Egg masses were monitored daily for the post hoc comparisons were performed using Tuckey's honestly sig- number of eggs hatched, and the time to the first hatching and the nificant differences (HSD) test (ANOVA for the plant samples with the percentage of hatching were determined. Hatching was considered to plant type as a factor and multivariate MANOVA for the snail samples have occurred when juvenile snails had emerged from both the egg and with the species/class and fraction as factors) to determine the differ- the egg mass. Observations were carried out for more than one month ences between the groups. Differences were considered to be significant until the snails had laid the masses and juveniles had hatched. Finally, at p ≤ 0.05 (STATISTICA 13.0 package Dell Inc., 2016). all the juvenile specimens were counted. The same procedure was fol- Multivariate analysis of the composition of was lowed for both types of masses (pond and laboratory). Hatching success performed using Canoco Ver. 4.5. Detrended Correspondence analysis was assessed for 29 field egg masses and 24 laboratory egg masses. (DCA) showed that the species responded linearly to the gradient (0.264 SD). The redundancy analysis (RDA) ordination technique was 2.3. Ecological study used to test for any significant linear trend in the community compo- sition and to develop a model to determine whether the measured ha- The field study of this research was carried out in an anthropogenic bitat variables were significant in explaining any variation in snail forest pond located in a mining area of southern Poland, 0.5 km from density. The Monte Carlo permutation was used for testing the sig- the Budryk coal mine (50°10′36.15″N, 18°46′04.60″E) to detect metal nificance of the model. Details of the application of RDA and the Monte bioaccumulation. The mining activity results in a land depression, Carlo permutation in model-ecosystem experiments can be found else- which after water filling leads to the creation of a new anthropogenic where (Van Wijngaarden et al., 1995; Makarenkov and Legendre, water body. In addition, the mining activity results in a high availability 2002). Twenty one explanatory variables were used in the model. Be- of metals. The study samples were collected from 2016 to 2017 (from fore applying the RDA model, the environmental data set and the May to November). To estimate the snail density, quantitative samples parameters were standardized to correct for the differences in the were taken from a bottom area of 0.5 m2. Because the density of measuring range between the different parameters, which would make freshwater snails differs in different sediments, sampling was done in them equally mathematically important. The axes of the RDA diagram two sites that differed in the type of substrate – allochthonous plant represent the percentage of the total sum of the canonical eigenvalues; matter (leaves from trees) and plant deposit (Typha latifolia remains). A thus, the higher these percentages, the better explained the observed square frame (0.25 × 0.25 m) was used for the quantitative sampling variation. According to Ter Braak (1988), values of about 30 –40% are (Maqboul et al., 2014). It was digged into the bottom four times for quite common in ecological applications. each sample. After taken to the laboratory, the samples of leaf and plant remains together with the snails were washed using sieves (0.02 mm 3. Results mesh size). The snails were sorted under a stereoscopic microscope and then preserved in 75% ethanol. We followed the identification method 3.1. Bioaccumulation study proposed by Glöer (2002) and Piechocki and Wawrzyniak-Wydrowska (2016). The snails were identified to the species rank based on their The BAF values of metals calculated for P. acuta differed in relation morphological features except - for the determination of to the size of the snail individuals (Table 1). The lowest BAF value of which we also included the pigmentation (Jackiewicz, 2000). The metals (except Zn) was found for the large class of specimens of this density of the snails was expressed as individuals per square meter snail species. The BAF value of cadmium decreased with the size of the (ind/m2). snails. For each size class and species the BAFCd and BAFPb were The most commonly measured ecological variables, including within the range of 0–2 and lower than the BAFCu and BAFZn, the community structure, and species diversity may reflect the stresses values of which exceeded 2. Irrespective of the size class and species, caused by the contaminants. Therefore, in the analysis of the gastropod the BAFCu was the highest among the BAF values of the analyzed community, we used the following indices: Dominance (D), the metals. Shannon -Wiener index (H′), and the Simpson diversity index (S) The analysis of the metal concentration in the fractions of snail (standardized version). The values of the Dominance index were con- individuals revealed that the contents of Cd, Pb, and Cu in the shell sidered within the ranges (four classes) according to Biesiadka and fraction were always significantly lower than in the body fraction Kowalik (1980). The diversity indices were calculated using MVSP (Fig. 1). The concentration of the metals in the shells was less than 20% (3.13.p Kovach Computing Services). of the concentration found in the body fraction. For the Zn concentra- The following parameters were analyzed in the field using a por- tion, a similar significant difference was only found in the group of table HI 9811-5 pH/EC/TDS/°C meter (Hanna Instruments, USA): con- ductivity (EC), total dissolved solids (TDS), temperature, and pH. Table 1 Analysis of the other parameters was performed in the laboratory (ti- Bioaccumulation factor values for the alien (P. acuta, P.a) and native (S. pa- trimetric determination) using the reagents from Merck and Hanna lustris, S.p.) snails. Instruments. Snail species, size metal 2.4. Statistical analysis Cd Pb Cu Zn

Statistical comparison among the physico chemical parameters of P.a. 5–7 mm (small) 1.35 1.73 7.39 2.45 the water and the densities of snails was performed using Student's t- P.a. 7.1–9 mm (medium) 0.73 1.87 9.43 1.85 – test. To compare the mean measurements obtained for the P. acuta P.a. 9.1 12.4 mm (large) 0.52 0.56 6.15 1.98 population, a one-way analysis of variance (ANOVA, F distribution) was S.p. 16–28.5 mm (large) 0.63 0.6 7.13 2.96 performed. The data were found to be of a normal distribution. Before

3 A. Spyra, et al. Ecotoxicology and Environmental Safety 185 (2019) 109703

Fig. 1. Metal concentration [μg/g dry weight] (mean ± SD) in the shell and body fractions of two snail species P. acuta (alien, three size classes) and S. palustris (native). Different letters (a, b) indicate statistically significant differences between the groups within fraction (ANOVA, Tukey's HSD test, p ≤ 0.05). medium-sized P. acuta snails. There were no significant inter- and in- the process of the incubation and hatching of the eggs of P. acuta traspecies differences in the metal concentrations in the shells. Except showed that after 5 days all the juvenile snails from 65% of the masses for Zn, the comparisons conducted within the size classes of P. acuta had hatched whereas after 14 days 100%. indicated that, in general, the lowest metal concentration in the body The masses deposited in laboratory conditions were very delicate, was typical for the largest individuals. The metal concentration in the transparent, and weaker than those deposited in the pond. There was body of the native species did not differ from the content measured in variation in their size with LM being slighty larger than the FM (mean their analogous group of the largest individuals of P. acuta (Fig. 1). length – 8988.59 μm) (Table 3). The average number of eggs was si- The concentrations of Cd and Zn in the Typha plant were sig- milar in the LM and FM, but a greater number of empty eggs was found nificantly higher than in the samples of leaves (Fig. 2). There were no in the LM. A 100% hatching success was observed in the egg masses significant differences in the concentrations of Cu and Pb between the collected in the pond (FM) but the snails from the masses laid the la- two sampled groups of plant materials. For all the metals studied, the boratory were hatched after seven days (94.8%). content in the sediments was higher compared to the organic matter Hatching success was not determined for S. palustris because the deposits (Table 2). collected specimens did not lay masses in any the replicate which we have done. Unfortunately, no egg masses of this species were found during the study period in the studied pond, and therefore, it was not 3.2. Experimental study possible to collect them.

Twenty nine egg masses of P. acuta were found in the studied pond (FM) (Table 3). The masses were attached to the plant remains, leaves, 3.3. Ecological study and other specimens of this species. They were transparent and de- posited in a thick, stiff, and relatively strong external casing. The length Water chemistry. The values of the physico-chemical parameters of of the masses reached up to 13,395 μm. After collection, they were the water at both sampling sites were similar except for ammonia found to contain from 29 to 77 eggs per cocoon. It was impossible to (t = −2.77, p < 0.05) and nitrates (t = −2.45, p < 0.05) (Table 4). determine precisely when the masses were deposited, but after trans- The pH of the water was high (7.8–8.6), and the values of the salinity ferring to the laboratory they began to hatch after five days. After 14 indicators were similar at both sampling sites. The water was soft with a days, all the eggs from the 29 masses had hatched (Table 3). Analysis of low content of phosphates, nitrites, and chlorides.

4 A. Spyra, et al. Ecotoxicology and Environmental Safety 185 (2019) 109703

Fig. 2. Metal concentration [μg/g dry weight] (mean ± SD) in the deposit of leaves and Typha. Different letters (a, b) indicate statistically significant differences between the groups (ANOVA, Tukey's HSD test, p ≤ 0.05).

Alien species in snail assemblages. P. acuta was the eudominant width). species (D > 10%) at both sampling sites (Table 2). The densities in the assemblages varied across the sampling sites for P. acuta (t = 3.44, 4. Discussion p < 0.05) but not for S. palustris (t = −0.83, p > 0.64). We found much higher densities of snails in the plant deposit but the values of the Freshwater ecosystems are known as the most valuable water re- diversity indices were almost similar at both sampling sites due to the sources which are affected by human influences. Pollution and de- presence of a very high number of one species - P. acuta. gradation of habitats have a negative effect on the water quality and The native S. palustris was found to be in the subrecedent class in the ecological conditions (Sahin and Zeybek, 2016); however, they also leaf deposit and the recedent class in the plant remains. Their density at contributes to the appearance of alien and invasive species. The alien P. both sampling sites was greater compared to other snails (Table 2). The acuta snails can efficiently disperse via water (Van de Meutter et al., diversity of snails was low at sampling sites due to the extreme abun- 2007) and have also been found to be carried with plant materials on dance of P. acuta. boats between lakes (Albrecht et al., 2009). According to Van Leeuwen RDA showed the significant relations of the snail communities with et al. (2013), the potential for aquatic snails such as P. acuta to exploit the environmental variables which were found to be statistically best several dispersal vectors may contribute to their wide distribution on explained by the variations (Fig. 3). The content of copper, zinc, and different continents and their successful invasion. This species inhabits ammonia was most strongly related to the patterns of distribution of the a broad range of waters and can survive, for example in sewage treat- alien P. acuta, and also of truncatula, Planorbarius corneus, Gyr- ment plants (Mácha, 1971; Hoffman, 1999; Turner and Montgomery, aulus crista, whereas in the case of native S. palustris was related to a 2009), and sewage drains and the associated puddles (Saha et al., higher concentration of calcium in the water (the first and second axes 2016). In the pond studied here, P. acuta was found to occur in high explain almost 40% (species data) and almost 99% (species and en- densities (maximal density of 5640 ind/m2) and as the dominant spe- vironmental relationships) of the variance, respectively). The Monte cies (D index near 94%) regardless of the substrate and sample; there- Carlo test showed that the model was statistically significant fore, it is likely that these features can be considered are responsible for (p < 0.05). Field samples showed that shell lengths differed among the its potential invasiveness. The environmental context cannot be dis- three selected age classes in the case of P. acuta (one-way ANOVA: regarded when investigating the interaction between the native and F = 150.17, p < 0.05 for shell height, F = 124.42, p < 0.05 for shell alien snails, and predicting the success and impact of the alien species

5 A. Spyra, et al. Ecotoxicology and Environmental Safety 185 (2019) 109703

Table 2 The composition of the snail communities, the content of heavy metals in the two types of habitats. Leaf deposit - the deposits of leaves fallen from the trees.

Leaf deposit Plant deposit

D [%] Mean density, D [%] Mean density

Physidae Physella acuta (Draparnaud, 1805) 94.1 931 ± 584.4 93.8 4003 ± 2102.2 Lymnaeidae balthica (L., 1758) 0.3 5 ± 9.2 0.0 0.0 R. auricularia (L., 1758) 0.6 19 ± 22.3 0.4 24 ± 34.9 S. palustris (O.F.Müller, 1774) 2.0 32 ± 29.6 1.7 75 ± 65.2 G. truncatula (O.F.Müller, 1774) 0.0 0.0 0.6 24 ± 41.6 Planorbidae G. crista (L., 1758) 1.9 19 ± 32.3 0.5 21 ± 12.2 P. corneus (L., 1758) 0.5 5 ± 4.6 0.2 11 ± 4.6 A. vortex (L., 1758) 0.0 0.0 0.1 5 ± 9.2 F. fragilis (Tryon, 1863) 0.8 8 ± 13.9 2.7 107 ± 184.8 Number of species in total 6 8 Total individuals 2968 12,808 Density of snails (individuals m−2) (mean) 989 4269 Density (individuals m−2) (min-max) 400–1624 1745–6187 Simpson diversity index 0.03–0.2 0.05–0.2 Shannon-Wiener diversity index 0.1–0.6 0.2–0.57 Content of heavy metals in deposits [μg/g] Mean (min-max) Cd 0.820 (0.06–2.75) 1.326 (0.56–2.42) Pb 10.367 (0.72–37.89) 12.574 (2.63–28.54) Cu 7.873 (0.86–19.47) 9.407 (4.89–15.20) Zn 83.742 (5.26–260.19) 150.552 (78.21–246.15) Content of heavy metals in the bottom sediments [μg/g]* Cd 3.28 Pb 80.48 Cu 28.16 Zn 499.62

Mean density is given ± 1SD (Standard deviation), * mean from the six measurements, D-dominance index, mean density is expressed in individuals/m.2.

Table 3 however, in their research the mean concentrations followed the se- Water chemistry at sampling sites. quence: Cu > Zn. In our study, the distribution of metals in both the

Leaf deposit Plant deposit body and the shell of P. acuta showed an opposite trend: Cu < Zn. It is likely that the authors of the above study analyzed the snails without Mean SD Mean SD assigning them to age groups. By contrast, our research showed that the uptake of the studied metals differ completely depending on the life Temperature ° C 20.3 2.7 21.0 3.2 stage of the snails. pH 8.6 0.5 7.8 0.5 fl Conductivity μS/cm 1138.7 344.5 1030.0 296.1 As was re ected by Boening (1997), Elder and Collins (1991), and TDS [mg/l] 566.7 180.1 610.0 195.2 Roméo et al. (2000), although the bioaccumulation of metals mainly Chlorides [mg/l] 138.0 54.1 148.7 92.9 depends on, for example the size, age, sex, feeding activity, and even Nitrates [mg/l] a 1.9 3.2 20.9 22.4 – genotype, it also relies on the environmental variables such as water N NO3 2.0 2,3 9.0 8.2 fi Nitrites [mg/l] 0.1 0.2 0.1 0.1 hardness, temperature, and pH as well as site-speci c conditions, which

N–NO2 0.02 0.006 0.02 0.01 according to Fischer (1983) and Wuncheng (1987), can moderate metal Ammonia [mg/l] a 0.3 0.2 0.7 0.2 toxicity considerably. Because both the studied species are grazers, Phosphates [mg/l] 0.8 1.2 0.1 0.1 which feed on macrophytes and epibenthic algae as a component of Total hardness mg CaCO /l 156.0 10.4 134.3 45.2 3 periphyton (Dillon et al., 2002), we aimed to compare their ability to Calcium [mg/l] 100.7 25.7 97.7 39.6 Iron [mg/l] 0.5 0.2 0.7 0.6 bioaccumulate metals because they uptake metals from their food Alkalinity 206.6 80.9 198.3 16.07 sources. Our study design did not allow us to assess or compare the extent of bioaccumulation and the BAF index in the three classes of the a fi - statistically signi cant (T-test, p < 0.05). native S. palustris as all specimens were large, and therefore, we com- pared the individuals of both species from one size class. We found that (Früh et al., 2017). the metal concentration in the body of the native species did not differ The accumulation of metals in aquatic invertebrates exposed to from the content measured in their analogous group of the largest in- pollution is well documented, and many ecological and ecophysiolo- dividuals of P. acuta. Analysis of metal uptake in different age classes gical studies suggest that molluscs respond to environmental stress and showed that the lowest metal concentration in the body was typical for pollution (Rainbow, 1995). Our study revealed that the content of Cd, the largest individuals, especially in case of Cd and Pb, but not Zn. In fi Pb, and Cu in the shell fraction was always signi cantly lower than in shells, this finding was similar, especially forthe accumulation of Cd the body of the snails studied. Bioaccumulation analysis of freshwater and Cu. The BAF value is often used for pollution biomonitoring snails is sometimes limited only to the soft tissues but their shell is also (Rosioru et al., 2016; Dirrigl et al., 2018). We found the lowest BAF important in accumulation, which can continue over the lifetime of the value for each metal in the P. acuta snails from large sixe class A distinct organisms (Imlay, 1982). In some cases, the accumulation of heavy decrease in the BAF values in relation to the size of the snails was found metals in shells can be substantial; however, some metals are accu- for cadmium (BAF = 0.52). Both species can be classified as macro- mulated more in the soft tissues than in the shells (e.g. lead in concentrators of Cu (BAF > 2). Lefcort et al. (2004) suggested that the (Radix) peregra)(Fischer, 1983). In contrast to our result, Karadede- ability of mining site snails to avoid heavy metals is due to both genetic Akin and Ünlü (2007) showed very low BAF values for Cu and Zn ac- and environmental factors, and snails are more sensitive to groups of cumulated in the body of P. acuta snails. They also did not detect Pb; metals than to individual metals. While Newman and Mcintosh (1982)

6 A. Spyra, et al. Ecotoxicology and Environmental Safety 185 (2019) 109703

Table 4 Characteristics of the egg masses and the process of eggs hatching and the characteristics of P. acuta masses deposited in the field (29 masses) and under laboratory conditions (24 masses).

FM LM

Length (μm) mean 9148.06 8988.59 min- max 7210.74–13,395.18 5028.35–14,093.09 Average number of eggs (min-max) 45 (29–119) 45 (13–106) Empty eggs 7 eggs from only one cocoon 45 eggs from 9 cocoons Hatching process day of collection after 5 days after 14 days after 20 days day of layout after 7 days

eggs per mass min – max 29–77 29–119 33–119 33–119 13–106 13–106 cocoons with all of the eggs hatched – 21of 29 29 29 – 19 of 24 Total number of eggs in masses 1313 1592 1686 1686 1095 1130 Total number of empty eggs ––––40 45 Average number of eggs in masses 45 54 54 58 45 46 + 4 empty eggs

FM - Field masses: masses deposited and collected from the pond, LM - laboratory masses: masses deposited in the laboratory (after 20 and until 30 days, 16 eggs still remain unhatched. pointed out that the concentrations of lead in molluscs reflected the decreasing tendency with an increase in the body weight of snails that degree of lead contamination at sites with different levels of Pb, they had originated from different biotopes. This confirm our results re- also concluded that the extent of habitat contamination only partially garding the bioaccumulation of Cd and Pb but not biogenic metals (Cu, helped in determining the lead concentrations in the molluscs. In the Zn), which in our study was also found decreased in the large speci- context of our study, the content of Cu and Zn in the substratum and mens. Heavy metal concentrations found in snails reflect the differences ammonia in the water was most strongly related to the patterns of in the extent of environmental pollution (Balogh et al., 1988); in our distribution of P. acuta whereas S. palustris was associated with a higher study, the metal concentration were always higher in plant deposits, concentration of calcium in the water. Balogh et al. (1988) found that especially Zn. the concentration of some toxic metals (Cr, Cd, Hg, Pb) showed a The ability to colonize anthropologically changed areas requires

Fig. 3. RDA biplot of snail species in relation to the strongest environmental variables. Thick arrows represent the environmental variables, and thin ones represent the snail species.

7 A. Spyra, et al. Ecotoxicology and Environmental Safety 185 (2019) 109703 efficient detoxifying or neutralizing mechanisms. The defensive me- Both storage in an inactive form and elimination in feces (Sá et al., chanisms against metal pollution include, among others, the elimina- 2008) or with exchanging gut epithelial cells (Pigino et al., 2006) re- tion of the excess of metals or the regulation of their concentration quire the initiation of the synthesis of additional metal-binding or en- through, for example, lower assimilation (Laskowski et al., 2010; zymatic proteins, which is energy-consuming. On the other hand, a Ardestani et al., 2014; Zhang and Van Gestel, 2017), or in the case of possible explanation for the relatively high BAF value in the youngest accumulation, their storage in an inactive form in intercellular granules individuals may be connected with an increased food consumption due or sequestration with ligands (e.g. metal-binding proteins) (Bebianno to the requirements connected with developmental processes compared and Langston, 1998; Wang and Rainbow, 2010). to the adult snails. Calculations of hatching success, however, indicate Stable populations are created by those species whose individuals that the reproductive functions in adult individuals were not disturbed. are characterized by either a wide tolerance to environmental pollution P. acuta is a hermaphrodite with the ability to self-fertilize (Raković or efficient neutralization or detoxification strategies. This efficiency of et al., 2016), and its development is direct, with no larval phase (Dillon a species to form a stale population depends on the trade-off and energy et al., 2002). Fertilization is internal, and the eggs are laid within a allocation between the parameters of life history and defensive me- gelatinous egg masses. The snails survive for one season and lay one chanisms (Jones and Hopkin, 1998; Augustyniak et al., 2005). These clutch of up to 50 eggs (Gittenberger et al., 2004). Its ability to self- phenomena seem especially interesting in the case of alien (and po- fertilize can further promote its rapid spread (Bousset et al., 2004). tentially invasive) species in anthropogenically polluted areas. Com- Analysis of the hatching of P. acuta showed a 100% hatching success parisons of the responses of the two mussel species (native: Anodonta after 14 days of incubation from the masses that had been laid in the anatine, exotic: Corbicula fluminea) to various mercury concentrations water body but hutching occurred after seven days from the masses laid under laboratory conditions revealed that the individuals of the in- under laboratory conditions (94.8%). Previous studies have docu- vasive species were less sensitive to this metal than the specimen of the mented that fecundity is not seriously influenced by the low con- native species (Oliveira et al., 2015). In this study the number of P. centrations of heavy metals (Ravera, 1991). At a high concentration of acuta individuals was much higher than the number of other native Cd, the fecundity of P. acuta is completely abolished, while it may be species in the snail community. depressed by lower concentrations such as 125 pg Cd/l (Agostini, Data concerning the metal body burden of P. acuta are extremely 1983). The hatching success and shell length of P. acuta decreased with scarce. Some information can be found for several metals in the study of an increase in Cu concentration, thus indicating the genetic toxicity Zaldibar et al. (2006). These data indicate that the metal concentrations effect of Cu (Gao et al., 2017), and also displaying a typical threshold in the soft tissues of P. acuta depend on the bioavailability of the metals response to increased salinity that was high - up to 5000 (Paradise, and suggest that these snails are able to cope with the environmental 2009). According to Ravera (1991), studies on the reproduction and pressure by accumulating metal ions in their body. embryonic development provide useful informations that can be used to It is suggested that the presence of stable populations of P. acuta in predict the survival probabilities of species living in polluted environ- spite of metal pollution is possible due to a kind of plastic adaptation ments. (Zaldibar et al., 2006), which is characteristic for the invasive features Summarizing, the ability of metal accumulation in the body does of an alien species (Davies et al., 2015), especially when it enables them not seem to be the factor promoting the invasive character of P. acuta. to dominate over the native ones. This phenomenon was experimentally Rather, its high reproductive potential (100% hatching success ob- confirmed in their study by Bielen et al. (2016), in which native and served from the egg masses collected in the pond) and ability to occur in alien species of bivalve were exposed to various zinc concentrations and high densities (mean 4003 ind/m2) enable this species invasiveness in temperatures under laboratory conditions. Comparisons of the bio- an anthropogenic aquatic ecosystems. marker levels revealed that the alien (invasive) species appeared to be more tolerant to both unfavorable factors (Bielen et al., 2016). In the 5. Concluding remarks present study, the comparisons of metal accumulating abilities between the native and alien aquatic snails were done under field conditions. This comprehensive study showed the high densities of the alien The concentrations Cd, Zn, Pb, and Cu in the body of the two snail snail species P. acuta in the studied sites. The content of ammonia, species did not differ significantly within their respective age classes. calcium, copper, and zinc were found to statistically best explain the Moreover, the BAF values for all the metals, except for Cu, were similar variations in the distribution of snails. The concentrations of Cd, Pb, in both species, which suggests that the individuals of both species and Cu in the shell fraction were always significantly lower than in the exhibit a similar strategy for metal neutralization: to some extent, the body fraction. The comparison of metal concentrations within the size elements were accumulated in their bodies but since the BAF values did classes of P. acuta indicated that the lowest concentration in the body not significantly exceed 2, it seems that both species they probably was typical for the largest individuals, except for Zn. Metal con- regulate the metal content at a similar level. As for Cu, the increased centration in the body of the native species did not differ from the body concentration and increased BAF value may be related to the fact content measured in the largest individuals of P. acuta. The lowest BAF that it is a biogenic metal and it increased accumulation may com- value for each metal was found in the large class of snails. A distinct pensate for a possible deficiency of this element in the environment. decrease in the BAF value in relation to the size of the snails was found Therefore, in anthropogenically changed environments, the environ- for cadmium. Moreover, the BAF values for all the metals, except for mental pressure does not seem to be a factor that promotes the occur- Cu, were similar in both species, which suggests that the individuals of rence of any of the species, unlike reported by Oliveira et al. (2015). both species represent a similar strategy for metal neutralization: to Since all the individuals of S. palustris in the collected samples were of some extent, the elements were accumulated in their bodies but since the largest (oldest) size class, individuals of the highest size class of P. the BAF values did not significantly exceed 2, it seems that both species acuta were included for the inter-species comparisons. In the intra- probably regulate the metal content at a similar level. Thus, the high species analyses conducted for P. acuta, the BAF values for all the reproductive potential (100% hatching success observed from the egg metals were in general higher in the youngest (smallest) snails com- masses collected in the pond) and ability to occur in high densities pared to the older (bigger) ones. However, opposite results were shown (mean 4003 ind/m2), rather than the ability of metal accumulation in in the study of Cubadda et al. (2001), which demonstrated a positive the body, seem to promote the invasiveness of P. acuta. correlation between the metal concentration and the body size of four marine gastropods. The higher BAF value found in young individuals Conflicts of interest may be regarded as representing a trade-off between a developmental and growth priority and an investment in metal elimination. The authors declare they have no conflict of interest.

8 A. Spyra, et al. Ecotoxicology and Environmental Safety 185 (2019) 109703

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